Optimization of ultrasonic Bath and cold plasma pre‐treatments in the spearmint essential oil isolation process

Abstract Spearmint essential oil (SEO), one of the economically valuable natural products, has special importance in the food, pharmaceutical, and perfumery industries due to its antifungal, antioxidant, and enzyme inhibitory properties. In this study, we optimized and evaluated the effect of three pre‐treatments on the extraction of SEO for quantity and quality: ultrasonic bath (UB), water to material ratio‐ultrasonic bath (W/M‐UB), and cold plasma‐ultrasonic bath (CP‐UB). Three experiments were designed using the central composite design (CCD) of response surface methodology (RSM). Experimental treatments included UB temperature (30–80°C) and time duration (1–30 min), cold plasma (CP) power (15–24 kV), and water/material ratio (10–40). Then, SEOs were extracted by hydro‐distillation using the Clevenger apparatus. The results showed that SEO yield in the optimal conditions of treatments was 119.7%, 206.6%, and 155.7% higher in UB, W/M‐UB, and CP‐UB pretreatments respectively, in comparison to control sample and optimized conditions were UB temperature: 37.3°C and UB time: 5.2 min at UB treatment, 33.9 of W/M ratio, 69.9°C of UB temperature and 6.9 min of UB time at W/M‐UB treatment and CP power: 22.176, UB temperature: 40.135 and UB time: 24.122 at CP‐UB treatment. Oxygenated monoterpenes were also higher in the essential oils (EOs) of all three treated plant materials. In conclusion, the SEO extraction yield improved by the application of the pretreatments in optimized conditions.


| INTRODUC TI ON
Medicinal plants are one of the main functional foods. A foodstuff is considered to be functional in addition to its nutritional properties when it has a positive effect on health, for example in illness prevention or cure. There are about 236 genera and more than 6000 species in the Lamiaceae family. Many species of the family are cultivated worldwide and most of them are aromatic plants. The mint family (Lamiaceae) is also one of the main functional food sources (Birch & Bonwick, 2019;Carović-StanKo et al., 2016).

Spearmint (Mentha spicata L.) is an annual plant from the
Lamiaceae family and is widely distributed in Africa, temperate Asia, and Europe (Kumar et al., 2011). Spearmint essential oil (SEO) is effective in the treatment of Alzheimer's disease, drug-resistant infections, dermatophytosis, and obesity (Ali-Shtayeh et al., 2019).
Worldwide, standardized SEO is one of the most useful additives in the food, pharmaceutical, cosmetic, and hygiene industries. In 2019, SEO (HS code: 330125) comprised of 20% of the total market value for essential oils (EOs; 1.3 billion dollars), whereas the market values for orange and peppermint EOs were 8% and 3%, respectively (Essential Oils (HS: 3301)) Product Trade, Exporters and Importers | OEC -The Observatory of Economic Complexity, (2020).
In recent years, various treatments were studied for EO extraction regarding their effect on chemical composition and the yield of EOs. One of the treatments evaluated by many researchers was the ultrasonic pretreatment (da Silva Moura et al., 2020;Liu et al., 2019;Priyadarshi et al., 2021;Zorga et al., 2020). Another attractive treatment experimented by a large number of researchers is the microwave pretreatment or microwave assisted extraction (Drinić et al., 2020;Radzi & Kasim, 2020;Tran et al., 2018) and etc. Other methods such as Ohmic-assisted hydro-distillation (HD; Gavahian et al., 2018), enzyme treatment (Morsy & Hammad, 2021), cold plasma (CP; Shokoohi et al., 2022), and salted distillation water treatment (Dao et al., 2020) were also evaluated in recent years for EO extraction from many medicinal and aromatic plants ( Table 1).
Ultrasonic pretreatment of EOs has recently attracted the attention of researchers. This technique can overcome some of the disadvantages of conventional HD. For example, the treatment can overcome the challenges of long extraction times and low-efficiency problems (Chen et al., 2007). HD can increase EO yields, as demonstrated in the following research works: Liu et al., 2019 (Iberis Amara seeds); Chen, Sun, et al., 2020 (Cinnamomum cassia bark); Boubechiche et al., 2017 (Allium sativum); Zorga et al., 2020 (Celery Seeds); and Chen, Liu, et al., 2020 (leaves of Perilla frutescens). In addition, the concentration of oxygenated compounds increase when this technique is used (Da Porto & Decorti, 2009). Carvone is one of the main components of SEO. The content of carvone (an oxygenated monoterpene) is increased under this treatment (Chen, Liu, et al., 2020;Damyeh et al., 2016). This component has a significant relation with the antioxidant activity (Younis & Beshir, 2004). The carvone to limonene ratio is one of the characteristics of SEO quality.
Studies have shown that the ratio of caraway seeds in cases of ultrasonic treatments was shorter than the control. (Assami et al., 2012).
Cold or non-thermal plasma has been used for its beneficial properties in agriculture. Enhancement of seed germination, decontamination, and soil remediation are some of these properties. Its efficiency for microbiological safety of plant and animal origin foods is also undeniable Ziuzina & Misra, 2016). The effect of dielectric barrier discharge cold plasma (DBD-CP) has been extensively studied for several types of EOs: In fennel seeds and spearmint leaves, the EO yield improved under the optimized DBD-CP treatment (Rezaei et al., 2021); in lemon peel oil extraction aided by DBD plasma treatment, the yield increased by 49.34%, although the TA B L E 1 Microwave, ultrasonic and Ohmic-assisted hydro-distillation and also cold plasma, enzyme and NaCl treatments in recently reported studies EO constituents remained constant (Pragna et al., 2019); also, camelina seed oil extraction by DBD-CP treatment showed a similar result. In general, studies have shown that the yield of oil extracted is positively affected by DBD-CP. The protein content obtained by this treatment was also the largest (Rezaei et al., 2020). These researches show the positive aspects of CP or DBD-CP application.
The above articles and research studies reveal the important effects of ultrasonic treatment and CP in addition to the varied performance of the extraction process in different water to plant ratios. To develop knowledge in this field, in this study, these treatments were tested together by RSM-designed experiments to determine their effects on the amount of extracted EOs. The optimal extraction conditions of EO as a result of the interaction of these factors are determined and the effect of these interactions on the composition of essential oils is also evaluated. To design these experiments, a CCD design of RSM was used.

| Experimental design
For the design of the experiment, the design expert software version 13 was used. The CCD method of RSM is commonly used in optimization experiments, including a full or fractional factorial design. A group of star points improved the center points of the design (Danh et al., 2009). In this study, the randomized CCD was utilized. The number of numeric factors in UB was two (UB temperature and processing time). In W/M-UB, three numeric factors were the W/M ratio, UB temperature, and UB processing time. In the third design, CP power, UB temperature, and UB processing time were the numeric factors.
The independent variables had five levels (

| UB pretreatment
The UB pretreatment was applied using an Elmasonic S 60 H UB processor (operating at 220-240 V Afterward, the treated samples were extracted by HD.

| Cold plasma procedure
The CP pretreatment was enforced using a dual power supply CP reactor (Marzdashty et al., 2017). Dimensions of the device were 70 (w) × 45 (d) × 60 (h) cm and reactor characteristics were as follows: power 1: 0-25 kV, 50 Hz; and power 2: 0-10 kV, 6 kHz. The device contained two parallel electrodes. One of these (the lower) was stainless steel and was connected to a glass bowl (quartz) to pro-

| Gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS)
Essential oil constituents were analyzed by gas chromatography The same GC oven temperature program was used.
The EO constituents were identified by matching each component's mass spectra with those of the internal mass spectra library of the main library, Wiley 7.0 and Adams. Further identification was based on a comparison of peak retention indexes by using a TA B L E 3 UB pre-treatment independent variables and experimental yield of CCD  (Adams, 2007).

| Effect of process factors on EO extraction efficiency
In this study, for UB design, the effects and interaction of UB temperature and time were studied. The interaction of the treatments with W/M ratio factors were subjected to W/M-UB design.
In the third experiment design, the CP power interaction with UB factors was the numeric factor. The CCD fixed each independ- was the obtained yield (Table 5).
For the experimental data of UB pretreatment, a 2FI model was fitted, and the following equation was obtained for the treated EOs yield, predicted R 2 = 0.7773 and adjusted R 2 = 0.9002: where Y is the EO percentage, A is the UB temperature, and B is the UB time.
For the experimental data of W/M-UB pretreatment, a linear model was fitted, and the following equation was obtained for the treated EOs yield, predicted R 2 = 0.8263 and adjusted R 2 = 0.8754: where Y is the EO percentage, A is the water to material ratio, B is the UB temperature, and C is the UB time.
Also for the experimental data of CP-UB pretreatment, a   (1) Y = + 0.5708 − 0.0891A + 0.0256B + 0.0483AB, Ultrasonic treatment has been shown to result in higher levels of EO extraction in cinnamon bark (Chen, Sun, et al., 2020), Iberis Amara seeds (Liu et al., 2019), citronella grass (Kumoro et al., 2021), and kumquat peel (Yu et al., 2021) in other studies. Figure 3 illustrates the EO percentage as a function of W/M-UB factors. From Figure 3, it can be understood that generally, the increase in W/M ratio leads to more extraction of EO after HD (Figure 3a,b). Figure 3c shows the effect of UB factors on EO percentage. From Figure 3c, it can be inferred that increasing UB temperature and decreasing UB time treatment improves SEO yield.

| Essential oil yield optimization as a function of W/M-UB factors interaction
The optimized conditions as a result of the interaction of the factors are 33.9 of W/M ratio, 69.9°C of UB temperature, and 6.9 min of UB time.

| Essential oil yield optimization as a function of CP-UB factors interaction
The optimization of EO extraction as a function of CP power, UB temperature, and UB time duration, to maximize the EO extraction efficiency was proven and was found to be as follows: CP power: 22.176, UB temperature: 40.135, and UB time: 24.122.
As shown in Figure 4a,b, a raise in CP power can improve the SEO yield after HD. As a result similar to other figures, UB temperature has an inverse effect on EO yield; however, UB time has a direct relationship with the SEO yield (Figures 3a-c and 4a-c). For example, in other studies, in lemon peel (Pragna et al., 2019) and cumin seeds (Sharanyakanth et al., 2021), CP treatment followed by HD has been shown to improve EO extraction.

| Effect of pretreatments on spearmint essential oil yield
Spearmint essential oil yield in the control sample (conventional HD) was 0.61%, whereas SEO percentages of 0.73%, 1.26%, and 0.95% were obtained from UB, W/M-UB, and CP-UB treatments. In other words, UB, W/M-UB, and CP-UB pretreatments in optimized conditions raised SEO yield to 119.7%, 206.6%, and 155.7% in comparison to the control sample ( Figure 6).

| Effect of pretreatments on EO composition
As shown in Table 7 and Figure 5, analysis of the EO resulted in the identification of 45 compounds. Oxygenated monoterpenes (from 71.07% in control to 77.52% in W/M-UB treated sample) represented 96%-98.32% of the total EO, followed by monoterpene hydrocarbons (from 7.46% in W/M-UB treated to 14.71% in control sample). This balance of the major components is in agreement with previous research (Rezaei et al., 2021). Carvone, limonene, dihydrocarveol <fneo->, cineole <1,8->, linalool, piperitenone, and menthone <iso-> were the main components in all samples.
In the optimized W/M-UB treatment, Menthone <iso-> content is significantly higher than control and other treated samples;  As shown in Figure 5, oxygenated monoterpenes yield in all treated samples was higher than control. On the other hand, the EO percentage in treated samples was higher than in the control ( Figure 6).  In addition, these treatments may improve antifungal and enzyme inhibitory properties. Further research and study of the economic aspects of these treatments on an industrial scale may help the EO and herbal medicine industries.

ACK N OWLED G M ENT
The authors gratefully acknowledge Mrs. Sanders for technical support.

FU N D I N G I N FO R M ATI O N
We are thankful to the Tarbiat Modares University (TMU) for funding this research.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are included in this published article.

E TH I C A L S TATEM ENT
"This study does not involve any human or animal testing".

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.